Fluorescent water-soluble perylenediimide-cored cationic dendrimers: Synthesis, optical properties, and cell uptake

Article abstract of DOI:10.1039/c3cc40330k

Different generations of perylenediimide-cored dendrimers with peripheral amine groups were synthesized. All these water-soluble dendrimers could rapidly internalize into live cells with high efficacy of gene transfection and low cytotoxicity. Increasing dendrimer generation increased their ability for gene transfection. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc40330k

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Fluorescent water-soluble perylenediimide-cored
cationic dendrimers: synthesis, optical properties, and
cell uptake†
Zejun Xu,a Bicheng He,b Jie Shen,b Wantai Yanga and Meizhen Yin*a
Cite this: Chem. Commun., 2013,
49, 3646
Received 15th January 2013,
Accepted 13th March 2013
DOI: 10.1039/c3cc40330k
www.rsc.org/chemcomm
Different generations of perylenediimide-cored dendrimers with microscopy. Degradable dendritic polyesters8 are quite attractive
peripheral amine groups were synthesized. All these water-soluble for biological applications. The peripheral primary amines serve
dendrimers could rapidly internalize into live cells with high as active sites for growing dendrons, and also provide positive
efficacy of gene transfection and low cytotoxicity. Increasing charges upon a final treatment with dilute hydrochloric acid. The
dendrimer generation increased their ability for gene transfection. chemical structures of three cationic dendrimers (G1, G2, and G3)
are shown in Scheme 1. Their optical properties in water were
Perylenediimides (PDIs) display exceptional chemical, thermal, and investigated. The cell-penetrating abilities of these dendrimers
photochemical stability with high fluorescence quantum yields in and the gene transfection efficacies were assayed using live cells
organic solvents. To date, PDI derivatives have been widely used in by fluorescent tracing of the complexes of dendrimers and DNA.
various fields, such as dye lasers, solar cells and photovoltaic
The synthesis strategy was started from amine-functionalized PDI
devices.1 However, due to the easy aggregation of perylene chromo- (1, Scheme S1 in the ESI†) that was prepared according to the
phores, PDIs exhibit poor water solubility and very weak fluores- literature procedure.9 The four primary amines in dye 1 selectively
cence in aqueous solution.2 This is the major challenge in the reacted with the acrylate group in 2-methacryloyloxyethyl acrylate
application of PDIs in biological areas. In order to increase their (MAEA) resulting in the intermediate product 2a.10 It should be
water solubility, modifications of perylene chromophores were noted that a slight excess of MAEA was needed to ensure the
achieved by directly incorporating hydrophilic moieties into the completion of the reaction. Repeated washing with hexane yielded
bay regions or imide positions.3 In general, the bay-substituted PDIs pure 2a. The methacrylates at the periphery of 2a selectively reacted
have an advantage of a significant red shift in the absorption with the thiol group in cysteamine (CA) resulting in 2b.11 The
maximum with a larger Stokes shift. This has attracted great interest purification of 2b was done by simply washing with cold brine to
as it minimizes the interference from cell auto-fluorescence.4 remove the unreacted CA. In this reaction dimethyl sulfoxide
Another modification strategy is the attachment of water-soluble (DMSO) could be added as an accelerator.12 The completion of this
star polymers5 or dendrimers to the PDI core. Dendrimers represent reaction was monitored using 1H NMR and MALDI-TOF MS spectro-
a class of perfectly branched polymers with a well-defined three- scopy. Fig. S1 (ESI†) shows that the characteristic peaks of the
dimensional shape. A characteristic of dendrimers is that numerous methylene group (CH2=) at 5.56 and 6.11 ppm disappeared com-
peripheral chain ends surround a single core.6 The previously pletely, indicating the completion of the ‘‘click’’ reaction of 2a. The
reported PDI-cored dendrimers were water-soluble, biocompatible, desired molecular weight of 3343.57 was observed using MALDI-TOF
and neutral with a nonionic character.7 Therefore, it would be of MS, confirming the successful synthesis of the first generation
high interest to incorporate ionic dendrons into the bay regions of dendrimer 2b. Similarly, alternately adding MAEA and CA produced
PDIs and explore their biological applications.
the second and third generation dendrimers (3b and 4b). In order to
In this study, we synthesized water-soluble PDI-cored cationic improve the water solubility and stability, the peripheral primary
dendrimers with different generations. The dendrimers were amines in the dendrimers (2b, 3b, and 4b) were treated with 2 M HCl
designed with three characteristics. The central fluorescent PDI which yielded their ammonium salts (G1, G2, and G3). The desired
chromophore allows the tracing of cell uptake via fluorescence products G1, G2, and G3 show good water solubility (>10 mg mLÀ1
)
and high photostability, which is essential for biological applications.
The life-time and sizes of these water-soluble dendrimers increased
with increasing generation (Tables S1 and S2, ESI†). The overall
synthesis strategy is shown in Schemes S1–S5 (ESI†). These
dendrimers were fully characterized using NMR spectroscopy
and MALDI-TOF mass spectrometry or gel permeation
a State Key Laboratory of Chemical Resource Engineering, Beijing University of
Chemical Technology, 100029 Beijing, China. E-mail: yinmz@mail.buct.edu.cn
b Department of Entomology, China Agricultural University, 100193 Beijing, China
† Electronic supplementary information (ESI) available: Synthesis procedures
and material characterizations. See DOI: 10.1039/c3cc40330k
c
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Fig. 1 The fluorescence images and intensities of G1, G2, and G3 internalizing
into cells.
increasing concentration. The detailed description and discussions
are provided in ESI.† The UV/Vis and fluorescence spectra of den-
drimers G1–G3 in aqueous solution showed that both the absorbance
and fluorescence intensities increased with the increase in dendron
generation (Fig. S3, ESI†). The maximum absorption and emission
was around 590 and 615 nm, respectively. The fluorescence quantum
yields (FQY) of these three desired dendrimers in water were deter-
mined. G1 showed a FQY of 9%. With increasing dendron generation,
there was a continuous increase in FQYs from 12% for G2 and up to
25% for G3. This showed that chromophore PDI was encapsulated in
the center and its aggregation in aqueous solution was noticeably
suppressed by the increase in the dendron generation.
To investigate the ability of these dendrimers to enter into live
cells, Drosophila S2 cells were incubated with these dendrimers.
Because all dendrimers contained a central PDI chromophore, their
distribution in live cells could be observed using fluorescence micro-
scopy. All dendrimers (G1, G2, and G3) were able to internalize into
cells (Fig. 1A–C). G2 and G3 were detectable inside the cells after 1 h
incubation at a concentration of 5 mM, and G1 required 2 h incuba-
tion, demonstrating the efficient and rapid internalization. As shown
in Fig. 1D, fluorescence intensities gradually increased in cells along
with the incubation time. The dendrimers with higher generations
were detected with stronger fluorescence intensities in cells. Similar
results have been observed in the literature13 in which fluorescein
labelled cationic PAMAM dendrimers were detected inside cells in an
increasing trend along with higher generation. Therefore, for such
macromolecular architectures, the dendrimers with higher genera-
tions have a stronger ability to be detected in cells. The greater
fluorescence intensity of the dendrimer with higher generation could
partially contribute to this. The molecular weight, shape and the
higher number of cationic groups on the surface have been inter-
preted as the causes for such difference in the cell uptake.13
Scheme 1 Chemical structures of three cationic dendrimers.
The above data demonstrated that all the three dendrimers could
be rapidly internalized into cells within short time incubation. The
positive charges are able to interact with negatively charged macro-
molecules such as DNA through electrostatic forces.14 In order to
assess whether these dendrimers (G1, G2, and G3) could act as
carriers binding DNA to internalize into cells, S2 live cells were
incubated with buffer containing dendrimer–DNA complexes at N/P
chromatography (GPC) analyses (Fig. S5–S22, ESI†). Detailed synthesis
procedures and material characterizations can be found in ESI.†
The concentration-dependent absorption and emission spectra of
dendrimers G1–G3 in aqueous solution are provided in Fig. S2 (ESI†).
Both the absorbance and fluorescence intensities increased with
c
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The highest cellular uptake ability and transfection efficacy of G3
was explained by bigger outer branches and more positive charges.
These dendrimers had remarkable gene transfection efficiency with
low cytotoxicity, and would be applied to live animals to explore the
potential gene therapy in ongoing experiments.
This work was financially supported by the National Science
Foundation of China (no. 21174012, 51103008 and 51221002),
Special Fund for Agro-scientific Research in the Public Interest
(no. 201003025), the 973 Program (2013CB127603), the
New Century Excellent Talents Award Program from Ministry
of Education of China (NCET-10-0215 and 09-0734) and
the Doctoral Program of Higher Education Research Fund
(20100010120006, 20120010110008).
Notes and references
Fig. 2 Gene transfection assay of dendrimers. Fluorescence images (A–C) of the
G3–DNA complex internalizing into cells after 48 h incubation (2 mM G3, 100 mM
DNA, N/P = 8 : 1). (A) G3 fluorescence image (red). (B) CXR Reference Dye labeled
DNA (blue). (C) Merged channels of (A) and (B). (D) Fluorescence intensities of
dendrimer–DNA complexes internalizing into cells after 48 h incubation (2 mM
dendrimer, 100 mM DNA, N/P = 8 : 1). The data were mean Æ SEM.
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In conclusion, the synthesis and optical properties of novel
water-soluble PDI-cored dendrimers bearing positive charges were
reported. The aggregation of the encapsulated PDI chromophores
was noticeably suppressed by outer cationic dendrimers, thus
leading to the enhancement of optical performances in water.
The fluorescence detection, water solubility, and low cytotoxicity,
together with biodegradability of polyester units, are the prime
attractions for bio-applications. All dendrimers could rapidly
internalize into live cells with high efficacy of gene transfection.
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This journal is The Royal Society of Chemistry 2013